The objective of our research for the past year is to characterize cytokine-stimulated adhesion receptor kinetics that enable endothelial cells to capture and guide neutrophils to the site of inflammation. The transition from random interactions of freely-flowing leukocytes with the endothelium to specific adhesion and diapedesis is a well-ordered mechanism with intercellular adhesion coordinated by signaling. E-selectin, for example, is not constitutively expressed on microvascular endothelial cells (MVEC), but is induced in response to cytokine or endotoxin stimulation. E-selectin supports neutrophil rolling, a preliminary step in leukocyte transmigration. While the temporal patterns of adhesion molecule expression in large vessel endothelium have been well characterized, there are fewer studies of MVEC, which is more closely related to the post-capillary venules where neutrophils transmigrate. We show that there is correlation between structural characteristics of E-selectin expression and functional adhesion (FA) between E-selectin and anti-E-selectin mAb-coated beads. MVEC were stimulated with IL-1a while two separate assays of E-selectin expression kinetics were performed for 4-6 hours using either flow cytometry or the micropipette FA assay. In flow cytometry measurements, the number of E-selectin receptors expressed on HMVEC increased 20-fold following 6 hours of stimulation. In parallel micropipette FA assays between HMVEC and anti-E-selectin mAb-coated beads, FA increased 10-fold. While FA depends on receptor and ligand density, parameters measured using flow cytometry, it is also determined by physical characteristics of surface contact intrinsic to the adhesion assay and effective receptor/ligand forward reaction rates. Ultimately, we plan to replace the antibody-coated bead probe with a neutrophil and study the kinetics of specific EC receptor- neutrophil ligand interactions. We have completed the studies on the static and dynamic lengths of neutrophil microvilli and this work has been published in the Proceeding of National Academy of Science. Microvilli, with most of the L-selectin and PSGL-1 (P-selectin glycoprotein ligand-1) on their tips, are believed to promote the initial arrest of neutrophils on endothelium. At the rolling stage following arrest, the lifetimes of the involved molecular bonds depend upon the pulling force imposed by the shear stress of blood flow. With two different methods, electron microscopy (with the cells fixed by a non-shrinking procedure) and micropipette manipulation, we have obtained two comparable neutrophil microvillus lengths, both about 0.3 5m in average. We have found also that, under a pulling force, a microvillus can be extended (microvillus extension) or a long thin membrane cylinder can be formed from it (tether formation). If the force is less than or equal to 34 pN (1 3 pN), the length of the microvillus will be extended; if the force is larger than 61 pN (1 5 pN), a tether will be formed from the microvillus at a constant velocity, which depends linearly upon the force. When the force is between 34 pN and 61 pN (transition zone), the degree of association between membrane and cytoskeleton in individual microvilli will dictate whether microvillus extension or tether formation occurs. When a microvillus is extended, it acts like a spring with a spring constant of 43 pN/5m. In contrast to a rigid or non-extendible microvillus, both microvillus extension and tether formation can decrease the pulling force imposed on the adhesive bonds, hence prolonging the persistence of the bonds at high physiological shear stresses. SIGNIFICANCE: This research will advance understanding of how circulating neutrophils effect phagocytosis.